2. REFERENTE TEÓRICO Y CONCEPTUAL
2.4 SABER Y DISCIPLINA ESCOLAR
Portions of this chapter appear in the following manuscript: expansion of breast
cancer stem cells with fibrous scaffolds.1
3.1 INTRODUCTION
Cells cultured in vitro two dimensional (2D) tissue culture plastics (TCP) lack of
the structural architecture necessary for proper cell-cell and cell-matrix interactions that
exists in vivo.2-4 The complexity of the in vivo system, being composed of many
uncontrollable factors, such as immune response, endogenous growth factors, hemodynamics and mechanical cues, complicates researches on effects of individual factor on cell behaviours. On the other hand, although rodent animal models are relative reliable
system for human cancer research and have been used for several decades,5-8 the significant
difference in telomerase regulation between humans and rodents makes transgenic and
inducible mouse cancer model questionable in term of replicating the human cancers.9
Hence, three-dimensional (3D) cell culture models emerged to bridge the gap between in
vivo human cancer studies and in vitro 2D cell culture models, as well as animal models.
The fabrications of 3D scaffolds for the study of tumor cells in vitro have adopted
techniques from developed fields of tissue engineering.2, 10-11 Among those techniques, the
electrospinning process is a simple and rapid method to generate scaffolds with fibers range
from micrometer to nanometer in diameter.12 Although cells in electrospun fibrous
scaffolds as a 3D-like culture system because cells are able to acquire the nutrition and
extracellular signal three dimensionally.13 Among various natural and synthetic polymers
available for electrospinning process, poly(ε-caprolactone) (PCL) have attracted interests of researchers due to low melting temperature, good blend-compatibility, FDA approval,
and low cost.14 Nano and micro scale fibrous scaffolds by electrospinning are ideal for 3D
cells culture, because their structures are similar to components in extracellular matrix,
providing essential cues for cellular survival, proliferation, organization, and function.13
Tumor cells grown in 3D scaffolds tend to develop the morphologies and
phenotypes observed in vivo15-17 and displayed higher invasive capability, and increased
drug resistance relative to cells in a 2D culture condition.18-19 However, the mechanism of
those alterations is still unclear. Recent researches revealed extracellular matrix (ECM) proteins can be either coated in 2D dishes or fabricate into 3D scaffolds for the
enhancement and maintenance of the stemness properties of cancer cells.20-21
Cancer stem cells (CSCs) are rare subpopulations that have the capability to initiate
tumor growth. Recent researches reveal that CSCs are responsible for metastasis,22 chemo-
resistance,23 and radio-resistance.24 In the present study, we hypothesized that the
electrospun PCL fibrous scaffolds itself without any protein component can increase CSCs
population. Using aldehyde dehydrogenase (ALDH) activity as a CSCs marker25 and
mammosphere formation assay,26 we found that MCF-7, T47D, SK-BR-3 and MDA-MB-
231 cells cultured in electrospun PCL fibrous scaffolds increased the CSCs population. The enhanced invasiveness and upregulation of epithelial-to-mesenchymal transition (EMT) markers indicates cells were undergoing EMT, which might trigger the transformation of
3.2 RESULTS AND DISCUSSION
3.2.1 Fabrication of Electrospun PCL Fibrous Scaffolds
PCL fibrous scaffolds were fabricated using a home-made electrospinning apparatus. For electrospun scaffolds to be reproduced, the important parameters of electrospinning were recorded and illustrated (Figure 3.1a). With a stationary collector placed at 10 cm underneath the tip, random fibrous scaffolds were obtained, and no preferred fiber orientation was observed under scanning electron microscope (SEM) (Figure 3.1b). The average fiber diameter calculated by Image J software was 1.63 ± 0.36 μm and the diameter distribution of this PCL scaffolds ranged from 0.76 to 2.35 μm (Figure 3.1c).
Figure 3.1. PCL fibrous scaffolds were fabricated by the electrospinning process. (a) The electrospinning setup was illustrated schematically. (b) The SEM image of the electrospun PCL fibrous scaffold. Scale bar indicates 100 μm. (c) The distribution of the fiber diameter on PCL fibrous scaffolds fabricated in the described setting.
Although in diseases or during the movement, the ECM will be reorganized in aligned orientation, the major component of ECM, collagen fibers, usually randomly surrounded the breast epithelial cells,28 which is similar to the organization of the electrospun PCL random fibrous scaffolds. Our lab found that luminal-type cell line MCF- 7 maintained cell-cell contact and random orientation on aligned fibrous scaffolds,
introduce the mechanical stretch on the cells, which might complicate the interpretation of results. Therefore, in this study we use random orientation fibrous scaffolds for cell culture.
3.2.2 Cellular Morphology and Growth on Electrospun PCL 3D Scaffolds
MCF-7 cells successfully propagated on PCL fibrous scaffolds. Fluorescence microscopy revealed different morphologies between MCF-7 cells cultured on electrospun PCL fibrous scaffolds and TCP (Figure 3.2). Cells on TCP exhibited trigonal or polygonal morphologies, whereas cells on PCL fibrous scaffolds showed round shuttle-like shape. We also observed similar results in T47D, SK-BR-3, and MDA-MB-231 cells (Figure 3.3). Relative to cells on TCP with a spreading-out morphology, cells on PCL fibrous scaffolds displayed smaller F-actin staining area and round-like shape. This is might be due to that fibers surrounding cells limited cells spreading and cells increased the thickness (Figure 3.4).
Figure 3.2. Fluorescence microscopy images of MCF-7 cells cultured in 2D tissue culture plastics. (a-c) and 3D PCL fibrous scaffolds (d-f). Blue indicates nuclei (DAPI); red indicates F-actin (Rhodamine-Phalloidin). All scale bars indicate 100 μm.
Figure 3.3. Fluorescence microscopy images of T47D, SK-BR-3, and MDA-MB-231 cells cultured in 2D tissue culture plastics and 3D PCL fibrous scaffolds . Blue indicates nuclei (DAPI); red indicates F-actin (Rhodamine-Phalloidin). All scale bars indicate 100 μm.
Figure 3.4. MCF-7 cells seeded in PCL fibrous scaffold occupied architectural features of the matrix in three dimensions as observed using confocal microscope. 3D reconstruction of sequential strata demonstrated MCF-7 occupation of sub-surface niches within the PCL scaffold. Serial z-plane sampling was conducted for total z-scan distance of 45 μm at 3 μm per plane for cells in PCL scaffolds (a) and 20 μm at 3 μm per plane for cells on TCP (b) . Blue indicates nuclei (DAPI); red indicates F-actin (Rhodamine-Phalloidin). All scale bars indicate 100 μm.
We utilized CellTiter-Blue assay measured cell growth on PCL scaffolds. On day 7, MCF-7 cells on TCP and fibrous scaffolds proliferated to 15.00 ± 0.48-fold and 13.87 ± 0.56-fold relative to day 1, respectively. There was no statistically significant difference in proliferation rate between cells on TCP and PCL fibrous scaffolds during the first 7 days. However, on day 9, cells cultured on TCP reached plateau, whereas cells on PCL fibrous scaffolds continue propagating to 17.58 ± 1.43-fold relative to day 1 (Figure 3.5). Considering the seeding cell densities were the same in these two culture systems, the discrepancy might come from the expanding space for cell growth in 3D PCL fibrous scaffolds. We also observed similar trends in MDA-MB-231 cells (Figure 3.6).
Figure 3.5. The proliferations of MCF-7 in 2D tissue culture plastics and 3D PCL scaffolds were measured at indicated time points by CellTiter-Blue assay. Results were shown as mean ± standard deviation. Statistical significance of the differences between cells on TCP and PCL is indicated by two asterisks (P < 0.005, n = 3).
Figure 3.6. The proliferations of MDA-MB-231 in 2D tissue culture plastics and 3D PCL fibrous scaffolds. Results were shown as mean ± standard deviation. Statistical significance of the differences between cells in TCP and PCL is indicated by two asterisks (P < 0.005, n = 3).
3.2.3 Culturing MCF-7 cells on PCL Fibrous Scaffolds Induces the Expansion of CSCs
The CSC theory postulates that a small group of cells, which is selected by specific combination of markers, are responsible for the initiation and maintenance of tumors. Chen and his colleagues found that MCF-7 cells in 3D collagen scaffolds displayed enhanced
stemness relative to counterparts in 2D culture.21 The collagen scaffolds provides not only a 3D culture scaffold, but also a microenvironment with protein-protein interactions between membrane proteins of MCF-7 cells and collagen. In this study, the unmodified electrospun PCL scaffolds excludes the protein-protein interaction between cells and scaffolds. We cultured MCF-7 cells on PCL fibrous scaffolds to test how this fibrous scaffolds culture condition affects the size of CSCs population.
Aldehyde dehydrogenase, which is a detoxifying enzyme responsible for the oxidation of intracellular aldehyde, may play a role in early differentiation of stem cells through its role in oxidizing retinol to retinoic acid.30 Accumulating data indicates that
ALDH expression is related to cells with enhanced tumorigenic and metastatic potential.31-
33 In addition, based on ALDH activity, Ginestier and colleagues successfully isolated
CSCs from human breast tumors, and the expression of ALDH1 was considered as a
predictor of clinical outcomes in breast cancer patients.25 We utilized the ALDEFLUOR
assay to assess the proportion of CSCs in breast cancer cells from PCL fibrous scaffolds
and TCP respectively.34 The ratio of ALDH-positive population of MCF-7 cells increased
to 6.33% ± 0.55% in 3D PCL scaffolds relative to 2.00% ± 0.04% in 2D TCP after 6 days culture without passage (Figure 3.7a and d). We also assessed CSCs in another two luminal-type cell lines, T47D and SK-BR-3, from PCL fibrous scaffolds and TCP. The proportion of ALDH-positive cells in T47D and SK-BR-3 increased to 6.80% ± 0.58% and 18.27% ± 1.73% on PCL fibrous scaffolds from 1.82% ± 0.74% and 7.22% ± 0.19% on TCP, respectively (Figure 3.7).
Figure 3.7. Epithelial breast cancer cell lines, MCF-7, T47D, and SK-BR-3 cultured on PCL fibrous scaffolds increased ALDH-positive population compared with counterparts cultured on TCP. (a-c) After being cultured 6 day on PCL and TCP without passage, cells were harvested and analyzed by ALDEFLUOR assay. The ALDH inhibitor, diethylaminobenzaldehyde (DEAB) (15 μM), was added to the cell suspension in
ALDEFLUOR assay buffer with the substrate as a negative control. Numbers indicate
percentage of cells in the gated area. (d-f) The percentage of ALDH-positive cells in MCF- 7, T47D, and SK-BR-3 cells cultured on PCL and TCP. Results were shown as mean ± standard deviation. Statistical significance is indicated by double asterisks (P < 0.001, n=3). Previous research reported that mammary epithelial stem and progenitor cells can survive and propagate in an attachment-independent manner and form floating spherical
colonies, which is termed mammospheres.26 Therefore the mammosphere formation assay
is often used to identify and enrich CSCs. To further confirm whether the PCL fibrous scaffolds culture system increases the size of CSCs population, we measured the mammosphere formation ability of MCF-7 cells from TCP and PCL fibrous scaffolds. We found the MCF-7 cells from PCL fibrous scaffolds formed approximately 2.0-fold more mammospheres than control cells from TCP (Figure 3.8a). The sphere formation efficiency
of T47D and SK-BR-3 from PCL fibrous scaffolds increased to 1.81% ± 1.07% and 3.44% ± 0.42%, relative to 1.07% ± 0.50% and 2.51% ± 0.16% for cells on TCP, respectively (Figure 3.8b-c). Some of those spheres have a large size of around 100 μm. These spheres
probably came from multiple cells fusion.35-36 Those data suggests that CSCs expanded in
those mammary cancer cell lines when cultured in electrospun PCL fibrous scaffolds.
Figure 3.8. Epithelial breast cancer cell lines, MCF7, T47D, and SK-BR-3 on PCL fibrous scaffolds increased the property of stemness. (a-c) Cells cultured on PCL and TCP for 6 days without passage were harvested for mammoshpere formation assay. Mammospheres were observed under microscopy and enumerated. Results were shown as mean ± standard deviation. Statistical significance is indicated by single asterisk (P < 0.05, n = 6) or double asterisks (P < 0.005, n = 6). Scale bars indicate 100 μm. (d) SOX2, SOX4, OCT3/4, and CD49f expression in MCF-7, T47D, and SK-BR-3 cultured on PCL and TCP. Results were shown as mean ± standard deviation. Statistical significance is indicated by single asterisk (P < 0.05, n = 3) or double asterisks (P < 0.005, n = 3).
OCT3/4 and SOX2 function cooperatively to regulate their own transcription and the transcription of a large set of other genes, which control the self-renewal and
pluripotency of stem cells.37 SOX4 functions in the progression of breast cancer by
orchestrating EMT.38 CD49f have been used for enrichment of normal and CSCs,39-40
which maintains the pluripotency and stemness through the direct regulation of OCT4 and SOX2.41 In our study, transcriptional factors related to stem cells, including SOX2 and OCT3/4, and breast CSC signatures, including SOX4 and CD49f, were enhanced in MCF- 7 cultured on fibrous scaffolds by 1.77 ± 0.25-fold, 2.22 ± 0.23-fold, 1.56 ± 0.25-fold, and 2.92 ± 0.33-fold, respectively, relative to cells on TCP (Figure 3.8a). In addition, among stemness related genes, only SOX2 were upregulated in T47D from PCL fibrous scaffolds,
while OCT3/4,SOX2, and CD49f were upregulated in SK-BR-3 cells from PCL fibrous
scaffolds, relative to cells from TCP (Figure 3.8b).
To investigate whether the effect of 3D PCL fibrous scaffolds culture on CSCs expansion is cell-phenotype specific, we examined how CSCs response to the fibrous scaffolds culture in high malignant basal-type cell line, MDA-MB-231 cells. ALDEFLUOR assay, mammosphere formation assay, and gene expression pattern all show that MDA-MB-231 cells on PCL fibrous scaffolds increased CSCs population relative to cells on TCP (Figure 3.9). Although the proportion of CSCs in MDA-MB-231 cells cultured on PCL fibrous scaffolds increases not as dramatically as in epithelial cell lines, the same trend of changes in the CSC population in several breast cancer cell lines cultured indicates that this phenomenon is not cell- phenotype specific.
Studies have demonstrated that tumor cells cultured in 3D systems displayed properties of increased malignancy and drug resistance compared to standard 2D cultured
tumor cells.17, 19, 42-43 Given the fact that CSCs are responsible for metastasis and drug resistance, the expansion of CSCs in tumor cells cultured in 3D may account for this change.
Figure 3.9. MDA-MB-231 cells cultured in PCL fibrous scaffolds displayed increased CSCs properties and gene expression pattern of EMT. (a) The percentage of ALDH- positive cells in MDA-MB-231 cells cultured in TCP and PCL Results were shown as mean ± standard deviation. Statistical significance is indicated by double asterisks (P < 0.001, n = 3). (b) MDA-MB-231 cultured in PCL and TCP were trypsinized and then plated in non- adherent conditions in mammosphere medium, as described in Methods. Mammospheres were numerated. Results were shown as mean ± standard deviation. Statistical significance is denoted by single asterisks (P < 0.05, n = 3). (c) qRT-PCR analysis was used to quantify the expression of stem cell markers, SOX2, SOX4, OCT3/4, and CD49f. (d) qRT-PCR analysis was used to quantify the expression of EMT markers in MDA-MB-231 from PCL and TCP. Results were shown as mean± standard deviation. Statistical significance is indicated by single asterisk (P < 0.05, n = 3) or double asterisks (P < 0.005, n = 3).
3.2.4 The Enhancement of EMT-Associated Properties in Cells from PCL Fibrous Scaffolds
The EMT is a developmental process, in which epithelial cells with tight cell junctions and relative low migratory ability convert to mesenchymal cells with migratory and invasive phenotype. Recent studies demonstrate that during the carcinoma progression,
some tumor cells are undergoing the EMT.44 The inducing factors of EMT include growth
factors, cell-matrix interaction, and hypoxia. In addition, EMT may also trigger the transformation of non-stem cancer cells to CSCs, in which tumor cells are acquiring
properties of invasion, drug-resistance, chemo-resistance, and radio-resistance.27
We previously reported that culture on PCL fibrous scaffolds with either aligned
fibers or random fibers can stimulate mouse mammary cell line H605 to undergo EMT.29
Here, we investigated whether human epithelial-type breast cancer cell lines MCF-7,
T47D, and SK-BR-3 cells cultured on PCL fibrousscaffolds were undergoing EMT.
Figure 3.10. Cells from PCL fibrous scaffolds culture displayed a mesenchymal morphology after being re-plated in TCP (right), relative cells from TCP (left). Scale bars indicate 50 μm.
A classical definition of EMT is a multiple step process, in which cells acquire a
7 cells from PCL fibrous scaffolds were reseeded to TCP, of which about 10% demonstrated elongated spindle-like morphology (Figure 3.10). There was no morphological change for reseeded T47D and SK-BR-3 cells from fibrous scaffolds. Using Transwell assay, we measured invasive property of MCF-7, T47D, and SK-BR-3 cells, a characteristic associated with EMT. Cells from PCL fibrous scaffolds showed significant increases in the number of cells passing through the membrane (Figure 3.11).
Figure 3.11. Culture on PCL fibrous scaffolds increases the invasion of MCF-7, T47D, and SK-BR-3 cells. (a-c) MCF-7, T47D, SK-BR-3 cells from PCL displayed higher invasive capability in Transwell invasion assay compared to counterparts from TCP. Cells that successfully migrated through the filter pores were fixed with 3.7% paraformaldehyde and stained with 0.5% crystal violet in 2% ethanol. Scale bars indicate 100 μm. (d-f) The average number of migrated cells per field was assessed by counting six random fields. Statistical significance is indicated by double asterisks (P < 0.001, n=3).
To further confirm cells on PCL fibrous scaffolds were undergoing EMT, we measured the transcriptions of EMT related genes in cells from two different culture conditions. The expression of EMT related transcriptional factors, including SLUG, SNAIL, TWIST1, TWIST2, ZEB1, ZEB2, and FOXC2, and other EMT markers, including VIM and ITGA5, were significantly upregulated in MCF-7 cells on PCL fibrous scaffolds
Figure 3.12. Immunofluorescence staining for E-cadherin in MCF-7 and T47D cells cultured in tissue culture plastics and PCL fibrous scaffolds. Blue indicates nuclei (DAPI);
red indicates E-cadherin. All scale bars indicate 100 μm.
upregulated, compared to cells on TCP. In SK-BR-3 cells, only ZEB2, VIM, and ITGA5
were upregulated. Meanwhile, the expression of the epithelial marker, CDH-1 (E-
cadherin), decreased in cells from PCL fibrous scaffolds relative to cells from TCP (Figure
3.14). Indirect immunofluorescence staining of cells with E-cadherin antibody revealed
3.12). Western blot analysis indicates there is no significant change in protein level for E- cadherin and vimentin in cells from fibrous scaffolds relative to cells from TCP (Figure 3.13). It is probably because the cells are still undergoing the early transition of EMT, which is a relatively slow process. For example, TGF-β normally induces EMT within
about two weeks.46 The improvement of invasion and enhancement of expression of EMT
markers suggests cells on PCL fibrous scaffolds were undergoing EMT.
Figure 3.13. After culturing for 6 days on PCL fibrous scaffolds and tissue culture plastics,
cell extracts were used for Western blot analysis to detect the expression of E-cadherin and
Vimentin. The density of each band was quantified with The Image J software and divided by the density of the corresponding GAPDH band. The ratio is presented under each blot after normalizing the values for TCP control as one unit.
It was reported that immortalized human mammary epithelial cells undergoing EMT displayed a shift in CD44 expression from variant isoforms (CD44v) to the standard
form (CD44s).47 In the present study, we observed CD44v, CD44s and CD44t were all
upregulated in cells cultured on PCL fibrous scaffolds (Figure 3.15). CD24 regulates TGF-
β3 and E-cadherin in oral epithelial cells during EMT.48 The downregulation of CD24 and
the upregulation of TGF-β3 in cells culture on fibrous scaffolds suggest cells were